August/13 August 2009

From 2009.igem.org

1.before Min prep
Checked the cell cultures transformed and plated out yesterday (8/10) for colony formation and made an approximate count of the number of colonies.

(plate number)-(location on plate) (no. of colonies)
                       1-2M                       100~    (Amp)
                       2-24G                      1?
                       1-12C                      10
                       1-6I                       10<
                       2-8I                       10<
                       2-6E                       10<
                       1-16G                      10
                       1-12A                      10<    
                       2-8O                       10< 
                       
                       2-11N                       10    (Kan)
                       1-18L                       50   

 inoculate to iquid medium(5mL + Amp 5uL or Kan 25uL)

after  ligation
              EpsE + terminator                    3        →  liquid medium
             RBS   +  LasR(2-8M)                 10     → rapid check



2.digestion and ligation

digestion with restriction enzyme

K204003

VectorInsert
RBS(1-2M)10cinR(1-14F)6
SpeI1XbaI1
PstI1PstI1
No.2 Buffer2No.22
dH2O6dH2O10
total20uLtotal20uL


K204004

VectorInsert
RBS(1-2M)10LasR(2-8M)10
EcoRI1EcoRI1
XbaI1SpeI1
No.22No.22
dH2O4dH2O6
total20uLtotal20uL


K204005

VectorInsert
1-18L81-23J8
SpeI1XbaI1
PstI1PstI1
No.2 2No.22
dH2O8dH2O8
total20uLtotal20uL


K204006

VectorInsert
RBS(1-2M)101-14D10
SpeI1XbaI1
PstI1PstI1
No.2 2No.22
dH2O6dH2O8
total20uLtotal20uL



37°C, 2hr


gel electrophoresis
Osaka090813.jpg

No. 3 , 4 , 5 , 6 , ladder

gel cut

purification by [QIAquick Nucleotide Removal Kit]

ligation

ligation
DNA        44
10* buffer 5
ligation   1
total      50uL
↓
16°C overnight



3.ligation check

3.1. EpsE + terminater

Result of Nanodrop concentration check
(sample No.) (concentration)
     2-A               21.5 ng/uL
     2-B               15.8 ng/uL
     2-C               16.7 ng/uL  


digestion with restriction enzyme


2-A/2-B/2-C8
XbaI1
PstI1
No.22
dH2O8
total20uL


1-23L(vector)8
XbaI1
No.22
dH2O9
total20uL


37°C over night


3.2. rapid check
Osaka090813-2.jpg

(RBS   +  LasR(2-8M))
No.3 and No.8 →  to iquid medium(5mL + Amp 5uL)


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